Determination of Fructose in the Presence of a Large Excess of Glucose

Some factors affecting the skatole-hydrochloric acid reaction for fructose were studied. Especially, the stability of the chromogen to light, the effect of the amount of chloroform for complete extraction of the chromogen from the aqueous phase, and the time course of color development at various temperatures were studied in some detail. The time course of color development in the β-indolylacetic acid-hydrochloric acid reaction for fructose was also investigated. Based on the results obtained from these observations, some modifications to both the skatole-hydrochloric acid and β-indolylacetic acid-hydrochloric acid reactions were proposed.

A modification of the resorcinol-thiourea-hydrochloric acid method of Roe et al.⁷⁾ for the determination of fructose is described. The modification is based on the observation that by lowering the incubation temperature to 70°C, a greater color intensity ratio (fructose color/glucose color) is obtained, thus increasing the specificity of the method for fructose. By applying the principle of the two-point determination of Mokrasch¹²⁾ to the modified procedure, fructose in the presence of 100-fold excess of glucose can be determined with an error of about 10%.

A modified procedure of the cysteine-carbazole reaction for the determination of fructose is described. By incubating the components of the color reaction at 40°C for 1 hr, fructose is determined with good sensitivity (the millimolar absorbance value of 29.3) and specificity (the color intensity ratio of fructose to glucose is about 240). When the principle of the two-point determination is applied to this modified procedure (1 hr and 3 hr at 40°C), fructose as small an amount as 0.4 μg in the presence of 250-fold excess of glucose can be determined with an error of about 10 %.     

Gene manipulation of catabolic activities for production of intermediates of various biphenyl compounds

Summary A bioconversion process was demonstrated by manipulation of catabolic genes. Catabolic intermediates of various biphenyl compounds could be efficiently produced by Pseudomonas aeruginosa carrying recombinant plasmids containing a set of cloned bph genes. A dihydrodiol compound was produced by the strain carrying plasmid pMFB4 containing bphA (encoding biphenyl dioxygenase) gene. A dihydroxy compound was produced from 4-chlorobiphenyl by the strain carrying plasmid pMFB6 containing bphA and bphB (encoding dihydrodiol dehydrogenase) genes. Tetrahydroxybiphenyl was accumulated as the final product via dihydroxybiphenyl from biphenyl by the same pMFB6 carrying strain. Meta-cleavage yellow compounds were produced from biphenyl and its derivatives substituted with methyl, chloro, bromo, or nitro group on one of the biphenyl rings by the strain carrying plasmid pMFB2 containing bphA, bphB and bphC (encoding dihydroxybiphenyl dioxygenase) genes.     

Burkholderia xenovorans LB400联苯双加氧酶及突变体对DDTs的降解

DDTs（dichlorodiphenyltrichloroethane,1,1,1-三氯-2,2-双氯苯基乙烷）是一种典型的持久性有机污染物,曾在疟疾防治和农业除虫方面被广泛应用。虽然包括我国在内的很多国家已经禁止使用DDTs,但目前对环境中DDTs的检测发现它仍然广泛存在且具有新的输入源。DDTs的持续存在对近海生态系统和人类健康具有一定危害,因此它所造成的环境污染问题仍然值得关注。由于Rieske型芳香羟化双加氧酶能够起始多种持久性污染物的降解,过去的几十年里一直是芳香化合物降解领域的焦点。[目的] 为探讨联苯双加氧酶对DDTs的降解特性及机制,本研究选取了食异生素伯克霍尔德氏菌LB400（Burkholderia xenovorans）联苯双加氧酶及突变体对p,p''-DDT和o,p''-DDT的降解过程进行研究。[方法] 以BphAE_LB400为亲本,通过两步定点突变将283位的丝氨酸突变为蛋氨酸,获得突变体BphAE_S283M。通过比较亲本酶与突变体对DDTs的催化性能,模拟突变蛋白结构和分子对接等方法,探究其降解特性及机制。[结果] BphAE_LB400和突变体BphAE_S283M都无法降解对位的p,p''-DDT,但突变体BphAE_S283M可以代谢o,p''-DDT并产生2个立体异构体。对接p,p''-DDT的BphAE_LB400和BphAE_S283M的结构分析表明,BphAE_LB400和BphAE_S283M中p,p''-DDT的反应环均不与原晶体结构中的联苯反应环重合。而对接o,p''-DDT的BphAE_S283M的结构分析表明o,p''-DDT的反应环与晶体结构中的联苯反应环距离很近,且2、3位的碳原子与单核铁原子催化中心的距离在0.5 nm以内,此外,BphAE_S283M的催化腔表面积和体积比BphAE_LB400更大,这很可能有助于BphAE_S283M与o,p''-DDT的结合。[结论] 283位氨基酸是影响BphAE_LB400对DDTs的催化代谢能力的关键氨基酸残基,它可以通过调节反应碳原子与催化中心的距离以及催化腔的大小来影响底物特异性。本次研究进一步阐明了283位氨基酸残基的影响机理,为更有效修复DDTs污染提供理论依据和技术支持。     

Study of biphenyl hydroxylase activity in Aspergillus parasiticus using a colorimetric assay

A colorimetric assay, based on the hydroxylation of 4-nitrobiphenyl (NBP), allows for the first time the quick measurement of the biphenyl hydroxylase activity of whole liquid cultures of Aspergillus parasiticus. The assay can be used to measure the activity in whole cultures containing biphenyl hydroxylase substrates other than NBP. The assay was used to investigate the magnitude and time dependence of the activity induced by different culture treatments. The biphenyl hydroxylase activities measured by the assay correlate with the rates of biphenyl substrate hydroxylation as determined by chromatographic (HPLC) analysis, so the assay is a convenient alternative to the HPLC method for determining effects of culture conditions on hydroxylation rates. The assay was used to demonstrate that biphenyl substrates (4-bromobiphenyl and m-terphenyl) and a product of biphenyl hydroxylation (4,4-dihydroxybiphenyl) induce (i.e., stimulate synthesis of) the hydroxylase system. The rates of hydroxylation of NBP observed in the assays (up to 0.6 g product/l per hour) are considerably higher than the rates of biphenyl substrate hydroxylation previously reported in cultures of A. parasiticus. Correspondence to: D. P. Mobley     

红球菌R04细胞的不对称分裂及其在联苯胁迫下的分裂抑制

【目的】研究红球菌R04细胞的分裂方式及联苯对其形态和细胞分裂的影响。【方法】以一株多氯联苯降解菌株(Rhodococcus sp.R04)为研究对象,利用荧光显微镜、扫描电子显微镜及透射电子显微镜分析红球菌R04在不同培养条件下的细胞分裂。【结果】红球菌R04细胞表现出对称分裂(约占30%)和不对称分裂(约占70%)两种分裂方式,且培养条件不影响不对称分裂细胞所占的比例。细胞分裂过程中,隔膜主要分布于细胞长度的30%–50%。在联苯的分解代谢过程中,红球菌R04细胞的生长分裂会受到联苯的抑制,但不影响红球菌R04细胞的分裂方式,在联苯胁迫下,细胞形成丝状化,表现出异常分裂,随着培养时间的延长,在细胞生长指数后期至转换期,细胞能够进行正常分裂。【结论】环境异生型化合物联苯/多氯联苯对其降解菌株——红球菌R04细胞的生长和分裂有较强影响,但是并不影响其分裂方式。     

A Reaction Product from Butylated Hydroxyanisole and Trimethylamine Oxide

The main reaction product obtained when butylated hydroxyanisole (BHA) and tri-methylamine oxide (TMAO) were thermally treated in liquid paraffin under a nitrogen stream at 180 C for 1 hr, was investigated. The crystalline substance obtained by the silica gel chromatography was recrystallized from ethanol and identified as the BHA dimer of biphenyltype, i.e., 2,2′-dihydroxy-5,5′-dimethoxy-3,3′-di-tert-butyl biphenyl, by means of the elementary analysis and the spectroscopic studies.

The BHA dimer was found to be inferior to BHA in the antioxidative activity which was compared according to the weight gain method, in either case when lard (at 60°C) and methyl esters of linseed oil (at 30°C) were used as substrates. The dimer showed a synergism with TMAO.     

Beijerinckia sp strain B1: a strain by any other name . . . . .

Beijerinckia sp strain B1 grows with biphenyl as its sole source of carbon and energy. A mutant, strain B8/36, oxidized biphenyl to cis-(2S,3R)-dihydroxy-l-phenylcyclohexa-4,6-diene (cis-biphenyl dihydrodiol). Strain B8/36 oxidized anthracene, phenanthrene, benz[a]anthracene and benzo[a]pyrene to cis-dihydrodiols. Other substrates oxidized to cis-dihydrodiols were dibenzofuran, dibenzothiophene and dibenzo-p-dioxin. Biphenyl dioxygenase activity was observed in cells of Beijerinckia B1 and B8/36 after growth in the presence of biphenyl, m-, p-xylene and salicylate. Recent studies have led to the reclassification of Beijerinckia B1 as Sphingomonas yanoikuyae strain B1. Subsequent biotransformation studies showed that S. yanoikuyae B8/36 oxidized chrysene to a bis-cis-diol with hydroxyl substituents at the 3,4- and 9,10-positions. Dihydronaphthalene was oxidized to cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, naphthalene, cis-1,2-dihydroxy-1,2-dihydronaphthalene and 2-hydroxy-1,2-dihydronaphthalene. Anisole and phenetole were oxidized to phenol. Thus the S. yanoikuyae biphenyl dioxygenase catalyzes cis-dihydroxylation, benzylic monohydroxylation, desaturation and dealkylation reactions. To date, the genes encoding biphenyl dioxygenase have not been cloned. However, the nucleotide sequence of a S. yanoikuyaeB1 DNA fragment contains five different α subunits as determined by conserved amino acids coordinating iron in a Rieske [2Fe-2S] center and mononuclear iron at the catalytic site. The specific role of the different putative oxygenases in biotransformation reactions catalyzed by S. yanoikuyae is not known and presents an exciting challenge for future studies. Received 29 May 1999/ Accepted in revised form 23 June 1999     

Organisation of the bph gene cluster of transposon Tn 4371, encoding enzymes for the degradation of biphenyl and 4-chlorobiphenyl compounds

  Tn4371 is a 55 kb transposon which encodes enzymes for the degradation of biphenyl and 4-chlorobiphenyl compounds into benzoate and 4-chlorobenzo-ate derivatives. We constructed a cosmid library of Tn4371 DNA. The bph genes involved in biphenyl/4-chlorobiphenyl degradation were found to be clustered in the middle of the transposon. Sequencing revealed an organisation of the bph genes similar to that previously found in Pseudomonas sp. KKS102, i.e. the bphEGF genes are located upstream of bphA1A2A3 and bphA4 is separated from bphA1A2A3 by bphBCD. Consensus sequences for σ54-associated RNA polymerase were found upstream of bphA1 and bphEGF. Plasmid RP4::Tn4371 was transferred into a mutant of Alcaligenes eutrophus H16 lacking σ54. In contrast to wild-type H16 exconjugants, the σ54 mutant exconjugants could not grow on biphenyl, indicating the dependence of Tn4371bph gene expression on σ54. The Tn4371-encoded bph pathway was activated when biphenyl and various biphenyl-like compounds were present in the growth medium. Preliminary observations indicate the presence of a region outside the catabolic genes downstream of bphA4 which is involved in mediating at least the basal expression of BphC. Received: 13 May 1996 / Accepted: 16 September 1996     

Dechlorination of polychlorinated biphenyl congeners by an anaerobic microbial consortium

  An anaerobic methanogenic microbial consortium, developed in a granular form, exhibited extensive dechlorination of defined polychlorinated biphenyl (PCB) congeners. A 2,3,4,5,6-pentachlorobiphenyl was dechlorinated to biphenyl via 2,3,4,6-tetrachlorobiphenyl, 2,4,6-trichlorobiphenyl, 2,4-dichlorobi-phenyl and 2-chlorobiphenyl (CB). Removal of chlorine atoms from all three positions of the biphenyl ring, i.e., ortho, meta and para, was observed during this reductive dechlorination process. Biphenyl was identified as one of the end-products of the reductive dechlorination by GC-MS. After 20 weeks, the concentrations of the dechlorination products 2,4,6-CB, 2,4-CB, 2-CB and biphenyl were 8.1, 41.2, 3.0 and 47.8 μM respectively, from an initial 105 μM 2,3,4,5,6-CB. The extent and pattern of the dechlorination were further confirmed by the dechlorination of lightly chlorinated congeners including 2-CB, 3-CB, 4-CB, 2,4-CB and 2,6-CB individually. This study indicates that the dechlorination of 2,3,4,5,6-CB to biphenyl is due to ortho, meta and para dechlorination by this anaerobic microbial consortium. Received: 30 April 1996 / Received revision: 26 July 1996 / Accepted: 5 August 1996     

Achieving structural diversity using the perpendicular conformation of alpha-substituted phenylcyclopropanes to mimic the bioactive conformation of ortho-substituted biphenyl P4 moieties: discovery of novel, highly potent inhibitors of Factor Xa

Ortho-substituted biphenyl moieties are widely used in drug design. We herein report a successful use of the perpendicular conformation of the alpha-substituted phenylcyclopropyl groups to mimic the aplanar, biologically active conformation of the ortho-substituted biphenyl moieties to achieve structural diversity. This is exemplified by the design and synthesis of a series of highly potent pyrazole bicyclic-based Factor Xa (FXa) inhibitors bearing alpha-substituted phenylcyclopropyl P4 moieties. The designed perpendicular conformation was confirmed by the X-ray structure of FXa-bound compound 2r. The potential structural basis for the high FXa potency in the phenylcyclopropyl P4 analogs and their improved FXa inhibitory activities compared with the biphenyl P4 counterparts are discussed.     

Catabolism of biphenyl by Pseudomonas sp. NCIB 10643 and Nocardia sp. NCIB 10503

Summary The metabolism of biphenyl by Pseudomonas sp. NCIB 10643 is reported in detail; that of Nocardia sp. NCIB 10503 is briefly investigated. Both organisms dissimilate biphenyl by the same route via oxidation to 2,3-dihydroxybiphenyl, meta cleavage to a product identified as 2-hydroxy-6-oxo-phenylhexa-2,4-dienoate which is then cleaved to give benzoate. Benzoate is a deadend metabolite in the pseudomonad but in the nocardia is further catabolised to catechol and thence to cis, cis-muconate. The enzymes involved in the individual steps of the proposed pathway have been assayed. The proposed pathway differs from that previously suggested for Pseudomonas sp. NCIB 10643 but is the same as found in other pseudomonads. This is the first report of catabolism of biphenyl in an actinomycete.     

Cis-2′, 3′-Dihydrodiol production on flavone B-Ring by Biphenyl Dioxygenase from Pseudomonas pseudoalcaligenes KF707 expressed in Escherichia coli

Escherichia coli JM109 strains expressing either toluene dioxygenase from Pseudomonas putida F1 or biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 were examined for their ability to catalyze flavones. Biphenyl dioxygenase produced metabolites from flavone and 5,7-dihydroxyflavone which were not found in the control experiments. The absorption maxima of UV-visible spectra for the metabolites from flavone and 5,7-dihydroxyflavone were found at 337 and 348 nm respectively by using a photodiode array detector in the HPLC. Liquid chromatography/mass spectroscopy (LC/MS) showed molecular weights 256 and 288 for the metabolites, respectively. The metabolite of flavone, which was isolated and purified from the bacterial culture, was further subjected to analysis by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. Based on the LC/MS and NMR results, biphenyl dioxygenase inserted oxygen at C2′ and C3′ on the B-ring of flavone, resulting in the formation of flavone cis-2′, 3′-dihydrodiol (2-[3,4-dihydroxy-1.5-cyclohexadienyl]-4H-chromen-4-one). Since this product is not found in Chemical Abstracts, this compound is considered a novel one. In addition, biotransformation of flavones by biphenyl dioxygenase suggested a potential role of bacterial dioxygenase to synthesize novel compounds from plant secondary metabolites. This revised version was published online in June 2006 with corrections to the Cover Date.     

Oxidative stress by biphenyl metabolites induces inhibition of bacterial cell separation

Cell–cell separation of a polychlorinated biphenyl (PCB)-degrading bacterium Comamonas testosteroni TK102 was monitored by flow cytometry. When monohydroxy metabolites of biphenyl (BP) (2-hydroxybiphenyl and 3-hydroxybiphenyl) were added to the culture, cell–cell separation of strain TK102 was inhibited at stationary phase. This inhibition was reproduced on non-PCB degrading bacteria such as Pseudomonas putida PpY101 and Escherichia coli MV1184, but was not observed on Pseudomonas aeruginosa PAO1. An opportunistic pathogen, P. aeruginosa PAO1, produces exopolysaccharide, which is known to scavenge damaging chemicals such as reactive oxygen species (ROS). The higher level of ROS and lipid peroxidants were detected in the cells treated by monohydroxybiphenyls. Fat-soluble vitamin E, which is a lipid radical scavenger, maintained bacterial cell separation during monohydroxybiphenyls treatment. Our results demonstrated that intracellular oxidative stress played an important role in the inhibition of bacterial cell separation during BP metabolism. This study shows that metabolites of environmental pollutants, such as monohydroxylated BP, inhibit bacterial cell separation by oxidative stress.     

Degradation of biphenyl by a Micrococcus species

Summary A bacterium capable of utilizing biphenyl as the sole source of carbon was isolated from soil and identified as a Micrococcus species. The organism also utilized 4-chlorobiphenyl and several other aromatic compounds as growth substrates. 2,3-Dihydroxybiphenyl and benzoic acid were identified as intermediates by physico-chemical methods. The bacterium degraded biphenyl to 2,3-dihydroxybiphenyl followed by its meta-ring cleavage to yield 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, which was then hydrolysed to give benzoic acid. Benzoate was further metabolised via a catechol meta-cleavage pathway by a Micrococcus sp.Correspondence to: H. Z. Ninnekar     

Growth inhibition of Rhodococcus sp. strain RHA1 in the course of PCB transformation

Summary Growth of a PCB degrader Rhodococcus sp. RHA1 on biphenyl and ethylbenzene was inhibited by 100 g/ml PCB 48. A PCB tolerant derivative of RHA1 designated RCD1 was deficient in growth on biphenyl. Southern hybridization experiments suggested that RCD1 has the bphDE gene deletion in a 390-kb linear plasmid of RHA1. The bphD gene complementation restored growth deficiency on biphenyl and growth inhibition on ethylbenzene by PCB 48, indicating that PCB metabolites are the cause of growth inhibition.     

Polychlorinated Biphenyl/Biphenyl Degrading Gene Clusters in Rhodococcus sp. K37, HA99, and TA431 Are Different from Well-Known bph Gene Clusters of Rhodococci

Four kinds of polychlorinated biphenyl (PCB)-degrading Rhodococcus sp. (TA421, TA431, HA99, and K37) have been isolated from termite ecosystem and under alkaline condition. The bph gene cluster involved in the degradation of PCB/biphenyl has been analyzed in strain TA421. This gene cluster was highly homologous to bph gene clusters in R. globerulus P6 and Rhodococcus sp. RHA1. In this study, we cloned and analyzed the bph gene cluster essential to PCB/biphenyl degradation from R. rhodochrous K37. The order of the genes and the sequence were different in K37 than in P6, RHA1, and TA421. The bphC8 _K37 gene was more homologous to the meta-cleavage enzyme involved in phenanthrene metabolism than bphC genes involved in biphenyl metabolism. Two other Rhodococcus strains (HA99 and TA431) had PCB/biphenyl degradation gene clusters similar to that in K37. These findings suggest that these bph gene clusters evolved separately from the well-known bph gene clusters of PCB/biphenyl degraders.     

Biphenyl synthase from yeast-extract-treated cell cultures of<Emphasis Type="Italic"> Sorbus aucuparia</Emphasis>

Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a subfamily of the economically important Rosaceae. The biphenyl aucuparin accumulated in Sorbus aucuparia L. cell cultures in response to yeast extract treatment. Incubation of cell-free extracts from challenged cell cultures with benzoyl-CoA and malonyl-CoA led to the formation of 3,5-dihydroxybiphenyl. This reaction was catalysed by a novel polyketide synthase, which will be named biphenyl synthase. The most efficient starter substrate for the enzyme was benzoyl-CoA. Relatively high activity was also observed with 2-hydroxybenzoyl-CoA but, instead of the corresponding biphenyl, the derailment product 2-hydroxybenzoyltriacetic acid lactone was formed.Abbreviations BIS biphenyl synthase - BPS benzophenone synthase - DTT dithiothreitol     

Oxidation of biphenyl by the Cyanobacterium,Oscillatoria sp., strain JCM

The oxidation of biphenyl by Cyanobacterium, Oscillatoria sp., strain JCM was studied. The organism grown photoautotrophically in the presence of biphenyl oxidized biphenyl to form 4-hydroxybiphenyl. The structure of the metabolite was elucidated by ultraviolet and mass spectra and shown to be identical to authentic 4-hydroxybiphenyl. In addition this metabolite had properties indentical to 4-hydroxybiphenyl when analyzed by thin-layer and high-pressure liquid chromatography. Experiments with [¹⁴C]-biphenyl showed that over a 24 h period the organism oxidized 2.9% of the added biphenyl to ethyl acetate-soluble products.Abbreviations tlc thin-layer chromatography - hplc high pressure liquid chromatography